Hem1^−/− DCs are impaired in T cell activation. (A) Percentage of activated T cells assessed by CD62L/CD69 surface expression at indicated OVA_323-339 peptide concentrations, three biological replicates, mean ± SD. (B) Exemplary CD62L/CD69 flow cytometry profile of T cells after 16 h of coculture with mature WT or hem1^−/− DCs. (C) IL-2 ELISA after 16 h of T cell mature WT or hem1^−/− DC coculture at indicated OVA_323-339 peptide concentrations, three biological replicates, mean ± SD. (D) CFSE dilution profile of T cells after 96 h of coculture with mature WT or hem1^−/− DCs at indicated OVA_323-339 peptide concentrations, representative example of three biological replicates. (E) Absolute T cell numbers after 96 h of coincubation with mature WT or hem1^−/− DCs at indicated OVA_323-339 peptide concentrations, three biological replicates, mean ± SD. (F) Proliferation indices of CFSE-labeled T cells after 96 h of coculture with mature WT or hem1^−/− DCs at indicated OVA_323-339 peptide concentrations, three biological replicates, mean ± SD. (G) Exemplary CD4/CD69 (top) and CD4/IL-2 (bottom) flow cytometry profile of T cells after 6 h coculture with WT or hem1^−/− DCs. (H) Fraction of IL-2–positive T cells after 4 or 6 h of coculture with mature WT or hem1^−/− DCs, pregated on CD4^high/CD69^high, two biological replicates, one-way ANOVA, mean + SD. (I) Normalized IL-2 mean fluorescence intensity (MFI) of CD4^high T cells after 4 or 6 h of coculture with mature WT or hem1^−/− DCs, two biological replicates, one-way ANOVA, mean + SD. Data were normalized to WT 4 h. 10 µg/ml brefeldin A was added for the last 3 h of the cocultures in G, H, and I. Data in A, C, E, and F were tested for normal distribution, transformed if necessary, and tested by using Student’s t test. ns, not significant. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.